The focus of this research is to understand how a novel member of the serpin superfamily, plasminogen activator inhibitor type 2 (PAI-2), protects immune cells from premature death and regulates the immune response. Macrophages are key host immune cells that reside in all tissues serving as sentinels to detect microbial invaders and other pathogens. Upon recognition of danger signals, macrophages produce cytokines, chemokines and other inflammatory mediators that alert the rest of the immune system. Progression of the immate immune response is dependent on macrophage survival, since danger signals may also trigger macrophage death under specific circumstances. Unless opposed, this can bring about rapid macrophage death and premature cessation of the innate immune response. To circumvent this, macrophages activate survival pathways coincident with proapoptotic pathways in order to control their life span and restrict the extent of their activation, which can be inherently self-damaging. Work from our laboratory and others has identified PAI-2 as a key inducible, cytoprotective factor that blocks the concurrent activation of a proapoptotic pathway. We found that PAI-2 stabilizes Rb and protects it from calpain cleavage, and in doing so, promotes Rb mediated activities associated with regulation of apoptotic signal transduction and proapoptotic E2F gene transcription. Based on the connections between PAI-2 cytoprotection and Rb anti-apoptotic activities, we initiated work on determining molecular mechanisms underlying PAI-2 cytoprotective activity, which has led to the identification of PAI-2 as an inhibitor of intracellular calpain. This competing renewal application proposes studies to determine the mechanisms involved in PAI-2 protection of Rb and E2F proapoptotic gene transcription and to determine the impact of this activity on macrophage immune surveillance and immune functions. The specific aims of the project are: 1) To investigate the molecular mechanism of PAI-2 inhibition of calpain cleavage of Rb, 2) To investigate the role of PAI-2 as a suppressor of macrophage mediated proinflammatory activity during innate immune responses, and 3) To characterize PAI-2 modulation of selective Rb repression of E2F regulated genes. The research plan focuses on biochemical analyses of molecular interactions, relevant cell culture systems, and models of innate immunity in adult mice. At the completion of these aims, we anticipate having established the molecular basis for PAI-2 mediated cytoprotection and its importance to inflammation and innate immunity